Part:BBa_K2220003

Display FliC(eGFP) protein

It can display flagellin fused with eGFP on the surface of S.cerevisiae.

Among various GPI-anchored proteins, α/a-agglutinins are one of the most common proteins have been utilized for the display of proteins on a yeast cell surface. The native a and α agglutinin receptors are believed to act as adhesion molecules to stabilize cell-cell interactions during mating and to facilitate fusion between the a and α haploid yeast cells. A-agglutinin has a core subunit encoded by AGA1 and a binding subunit encoded by AGA2. Both a-agglutinin and the core subunit of a-agglutinin consist of a secretion-signal region, a functional region, a support region, and a putative GPI anchor-attachment signal. After genetic engineering, foreign protein can be fused to the C-terminal of the binding subunit of a-agglutinin. As AGA1 protein is secreted from the cell and becomes covalently attached to the β-glucan in the extracellular matrix of the yeast cell wall, aga2 protein fused with foreign protein on its C-terminus will then bind to aga1p through two disulfides bonds. As a result, the heterologous proteins are covalently linked with glucan-layer.

The flagellar filament is a helical structure composed of subunits of flagellin. And among various flagellin, the FilC has its own unique feature. The N- and C-terminal region of the FliC are important for filament formation, which are the D0 and D1 domain. While the D3 domain which is a central region of the flic playing no role in filament formation. Additionally, when FliC proteins are in the polymeric state, the D3 domain can be exposed on to the surface. So from above, we can replace the central region of flagellin by other proteins without destroying self-assembling ability and if we fuse the enzyme and FliC, we can obtain a filamentous material with high surface density of catalytic sites.

We use AGA2 gene to replace the central region of flagellin. So that the part can display flagelin on the surface of S.cerevisiae.And we add the eGFP gene to the C-terminal of AGA2 to detect this process.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1770
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 301
  
• 1000
  COMPATIBLE WITH RFC[1000]